Those DNA regions of a gene which are located upstream of the transcription initiation point and by which the initiation point and the initiation frequency of the transcription and thus the expression level and the expression pattern of the controlled gene are determined, are in general referred to as promoters. RNA polymerase and specific transcription factors activating the RNA polymerase bind to the promoters in order to initiate transcription together with the basal transcription complex. The effectiveness of the promoters is often enhanced and regulated by additional DNA sequences, the enhancer sequences, whose position, contrarily to the position of the promoters, is not fixed. These regulatory elements can be located upstream, downstream, or in an intron of the gene to be expressed.
In recombinant DNA technology, promoters are inserted into expression vectors in order to control the expression of a transgene, which is normally not the gene naturally regulated by the promoter. Of substantial significance herein is the specificity of the promoter, which determines at which point in time, in which types of tissues, and at which intensity a gene transferred by means of genetic engineering is expressed.
In plant breeding, recombinant DNA technology is often used for conferring specific advantageous properties to useful plants, which is supposed to lead to a higher yield, for example by means of increased pathogen resistance, or to improved properties of the harvest products. Herein, it is often desirable that the transferred gene be not expressed ubiquitously, but only in those tissues, where the transgenic activity is desired, as the presence of the transgenic product can have a negative effect on normal physiological processes in some tissues. Thus, it could, for example, be shown that the overexpression of an anionic peroxidase under the control of the 35S promoter leads to wilting of transgenic tobacco plants, as less root growth occurs and therefore also less root mass is developed (Lagrimini et al. (1997) Plant Mol Biol. 33 (5), S. 887-895). The overexpression of the spi 2 peroxidase under the control of the ubiquitin promoter leads to reduced epicotyl development and reduced longitudinal growth in comparison with control plants (Elfstrand, M. et al. (2001) Plant Cell Reports 20 (7), S. 596-603). Irrespective of negative effects on physiological processes, it is often supposed to be prevented in resistance breeding that the transgenic product is also present in the harvested plant parts.
Therefore, promoters functioning either tissue-specifically or inducibly have been isolated during the past years. Tissue-specific promoters are, for example, seed-, tuber-, and fruit-specific promoters. The inducible promoters can be activated, for example, by means of chemical induction, light induction, or other stimuli.
It is also desirable to specifically modulate gene expression in the epidermis. The epidermis is the terminal tissue of the above-ground organs of higher plants. As such, the tasks of the epidermis are, on the one hand, to allow water and nutrient exchange of the plant and, on the other hand, to prevent the intrusion of pathogens into the plant. These functions can be specifically modulated by means of altered gene expression in the epidermis with the aid of suitable promoters and genes controlled by the latter.
Epidermis-specific promoters have already been described in dicotyledonous plants. It could thus be shown that the promoter of the CER6-(CUT1-) gene from Arabidopsis, which codes for a condensing enzyme in wax synthesis, can cause the epidermis-specific expression of a β-glucuronidase reporter gene (Hooker et al. (2002) Plant Physiol. 129(4): 1568-1580; Kunst et al. (2000) Biochem. Soc. Trans. 28(6): 651-654).
However, only few suitable epidermis-specific promoters in monocotyledonous plants, which are particularly well suitable for the expression of transgenes in monocotyledons, in particular in poaceae (sweet grasses), have been identified up to now. Recently, a promoter has been described which is composed of the promoter of the GSTA1 gene and the intron of the WIR1a gene and has a constitutive epidermis-specific activity (DE 103 46 611 A1).
As only few suitable promoters for epidermis-specific transgenic expression have been known hitherto, constitutive promoters like the ubiquitin promoter from maize were mostly used in order to express proteins in the epidermis (see, for example, Oldach et al. (2001): Mol Plant Microbe Interact. 14(7): 832-838). However, this can lead to undesired side effects in the transgenic plants due to the presence of the transgenic product in tissues or organs other than the epidermis, as is described above.
It is therefore a problem underlying the present invention to provide means allowing an epidermis-specific and pathogen-inducible gene expression in monocotyledons, preferably in cereal plants.
This problem is solved by providing the embodiments characterized in the patent claims.